ENZYMOLOGY OF GOLGI STACK FORMATION

我们发现了一种能使高尔基体起水泡的新型化学物质--利马喹酮。 膜和解聚微管，而其他细胞内 细胞器和细胞骨架元素不受影响。在移除时 智商，微管和高尔基复合体都会迅速重组， 它们的原始形态。在此组装过程中，VGM(用于发泡的 高尔基体膜)首先融合形成成堆的高尔基体池。这 这一过程不需要微管，也可以在16 而不是在低于16摄氏度的温度下。 微管的存在和37℃下的孵化聚集为 中央中央周围区域的复合体。我们已经重组了议会 半完整细胞中来自VGM的高尔基堆叠。此堆栈组件具有 对ATP、GTP的水解酶、在16-37℃孵化的必备要求 摄氏度和胞浆蛋白。我们有一个独特的机会来孤立 催化VGM之间融合的蛋白质并提供 用于逐步组装高尔基堆栈的结构脚手架。 我们想要描述堆叠组装过程中的中间体，以及 定义在功能上处于活动状态的最小装配结构 囊泡蛋白运输。我们将分离胞质GAF(用于高尔基体 装配系数)将VGM组装成堆栈所需的 水池。我们想要分离gafs有丝分裂的对应物以进行测试。 这些蛋白质是否经过任何翻译后修饰，如 与细胞周期事件相关的磷酸化/去磷酸化。 我们想要解决的问题是，这些翻译后 修饰具有功能意义，酶是什么？ 负责调节细胞周期中的这些修饰。 因此，将一组纯化的gafs、抗体和cDNA结合在一起 通过功能分析，我们想要阐明堆叠的机制 的高尔基体池维持在间期细胞中，并建立在每个 细胞分裂后的子细胞。这是以前从未尝试过的， 因此有望揭示参与调节细胞周期的新蛋白质 高尔基堆叠的立体组织及其意义 这种组织在囊泡蛋白的运输中。

We have identified a novel chemical ilimaquinone that vesiculates Golgi membranes and depolymerizes microtubules, while other intracellular organelles and cytoskeletal elements remain unaffected. Upon removal of IQ, both the microtubules and the Golgi complex rapidly reassemble to their original form. During this assembly process VGMs (for vesiculated Golgi membranes) first fuse to forms stacks of Golgi cisternae. This process does not require microtubules and can also take place at 16 degrees C but not at temperatures below 16 degrees C. The stacks then in the presence of microtubules and incubation at 37 degrees C congregate as a complex in the pericentriolar region. We have reconstituted the assembly of Golgi stacks from VGMs in semi-intact cells. This stack assembly has an obligate requirement for ATP, hydrolysis of GTP, incubation at 16-37 degrees C and cytosolic proteins. We have a unique opportunity to isolate the proteins that catalyze the fusion between VGMs and provide a structural scaffold for the step wise assembly of Golgi stacks. We want to characterize intermediates in the stack assembly process and define the minimum assembled structure that is functionally active in the vesicular protein transport. We will Isolate cytosolic GAFs (for Golgi Assembly Factors) required for the assembly of VGMs into stacks of cisternae. We want to isolate the mitotic counterparts of GAFs to test whether these proteins undergo any posttranslational modifications such as phosphorylation/dephosphorylation in association with cell-cycle events. The questions we want to address are whether these posttranslational modifications have a functional significance and what are the enzymes responsible for the regulation of these modifications during cell-cycle. Therefore, with a battery of purified GAFs, antibodies and cDNAs combined with a functional assay we want to elucidate the mechanism by which stacks of Golgi cisternae are maintained in interphase cells and built in each daughter cell after cell-division. This has never been attempted before, and therefore promises to reveal novel proteins involved in regulating the three dimensional organization of Golgi stacks and the significance of this organization in vesicular protein transport.